摘要
目的:构建Smads转录激活检测系统,以研究Smad2、Smad3、Smad4的功能。方法:将Smad2/3/4编码序列以正确读码框与pRC-lac载体中的Lac阻遏蛋白编码序列融合构建重组质粒,将重组质粒与受Lac操纵子调控的荧光素酶报告基因质粒共转染293T细胞,检测荧光素酶活性,其活性的强弱即代表转录活性的强弱。结果:Smad2和Smad3具有较强的依赖于TGF-β1的转录活性;Smad4全长没有检测到转录活性,但其SAD结构域有不依赖于TGF-β1的转录活性,MH2结构域有依赖于TGF-β1的转录活性。结论:构建的Smads转录激活检测系统能有效地用于Smad2、Smad3、Smad4功能的研究。
Objective： To construct Smads transcriptional activation system for studying the function of Smads. Method： The corresponding cDNA coding sequence of Smad2/3/4 was inserted to the pRC-lac vector which contains Lac repressor coding sequence. The recombinants were transfected into 293T cells with plac-LUC reporter, and then the luciferase activity was detected. Results： Smad2 and Smad3 had TGF-β1-induced transcriptional activity. Full-length Smad4 did not have transcriptional activity, but Smad4 SAD and MH2 domains had TGF-β1-independent and TGF-β1-dependent transcriptional activities, respectively. Conclusion： Smads transcriptional activation system was successfully constructed. This will provide a good tool for studying on function of TGF-β1 signaling pathway.
出处
《生物技术通讯》
CAS
2007年第2期189-192,共4页
Letters in Biotechnology
基金
国家自然科学基金项目(30625035
30530320
30470378)
国家重点基础研究发展规划项目(2006CB0F0300)
